Plasmids are small, circular molecules of DNA that exist separately from the main chromosomal DNA within a cell. These genetic structures are capable of self-replication, meaning they can make copies of themselves independently. While often associated with simpler life forms, plasmids are increasingly used in the study and manipulation of complex eukaryotic organisms. They serve as tools to introduce and express specific genetic information within eukaryotic cells.
Plasmids in Prokaryotes Versus Eukaryotes
Plasmids are abundant and diverse in prokaryotic cells, such as bacteria and archaea. In these organisms, plasmids frequently carry genes that provide advantages, like resistance to antibiotics or traits that enhance their ability to cause disease. These plasmids can be readily transferred between prokaryotic cells, contributing to their adaptability and evolution.
However, eukaryotic cells, which include animals, plants, and fungi, generally do not naturally contain independent, self-replicating plasmids. The absence of naturally occurring plasmids in eukaryotes means scientists must engineer them specifically for use in these complex biological systems.
Engineering Plasmids for Eukaryotic Systems
Given their natural absence, scientists engineer plasmids to function within eukaryotic cells.
Eukaryotic Promoter
A primary component added is a eukaryotic promoter. This acts as a “start” signal, directing the cell’s machinery to begin transcribing the gene of interest into RNA. Without a proper eukaryotic promoter, the inserted gene would not be expressed.
Origin of Replication and Polyadenylation Signal
Another necessary addition is a eukaryotic origin of replication, a specific DNA sequence that allows the plasmid to be copied within the host cell. A polyadenylation signal is also included at the end of the gene sequence. This signals the cell to add a string of adenine nucleotides to the messenger RNA, which stabilizes it and aids in its transport out of the nucleus for protein synthesis.
Selection Marker and Shuttle Vectors
Engineered plasmids incorporate a selection marker, such as a gene conferring resistance to an antibiotic like G418 or puromycin. This marker allows researchers to identify and select only those eukaryotic cells that have successfully taken up and maintained the plasmid, as only these cells will survive in the presence of the antibiotic. Many engineered plasmids are designed as “shuttle vectors.” These contain origins of replication and selection markers for both bacterial and eukaryotic systems, allowing them to be easily manipulated and amplified in bacteria before being introduced into eukaryotic cells.
Applications of Plasmids in Eukaryotic Research and Medicine
Engineered plasmids have become important tools in many eukaryotic research and medical applications.
- Gene Expression: Plasmids prompt eukaryotic cells to produce specific proteins. For example, plasmids can carry genes for human insulin or therapeutic antibodies, enabling large-scale production of these proteins for medical use.
- Gene Therapy: Plasmids are also used in gene therapy, delivering therapeutic genes into patient cells to correct genetic defects or treat diseases. A plasmid carrying a functional gene copy can be introduced into cells lacking that gene, potentially restoring normal cellular function.
- Gene Editing: Plasmids serve as a delivery vehicle for components of gene-editing systems, such as CRISPR/Cas9. Plasmids can carry genes encoding the Cas9 enzyme and guide RNA sequences, enabling precise modifications to the host cell’s genome for research or therapeutic purposes.
- Vaccine Development: In vaccine development, plasmids are used as DNA vaccines, containing genes encoding specific antigens from a pathogen. When introduced into the body, host cells express these antigens, stimulating an immune response without exposing the individual to the actual pathogen.
- Basic Research: Beyond these applications, plasmids are employed in basic research to investigate gene function, study protein localization, and unravel complex cellular pathways, providing insights into biological processes.